Growth Retardation of Poorly Transfectable Tumor by Multiple Injections of Plasmids Encoding PE40 Based Targeted Toxin Complexed with Polyethylenimine

Background:: One of the approaches to cancer gene therapy relies on tumor transfection with DNA encoding toxins under the control of tumor-specific promoters. Methods:: Here, we used DNA plasmids encoding very potent anti-ERBB2 targeted toxin, driven by the human telomerase promoter or by the ubiquitous CAG promoter (pTERT-ETA and pCAG-ETA) and linear polyethylenimine to target cancer cells. Results:: We showed that the selectivity of cancer cell killing by the pTERT-ETA plasmid is highly dependent upon the method of preparation of DNA-polyethylenimine complexes. After adjustment of complex preparation protocol, cell lines with high activity of telomerase promoter can be selectively killed by transfection with the pTERT-ETA plasmid. We also showed that cells transfected with pTERT-ETA and pCAG-ETA plasmids do not exert any detectable bystander effect in vitro. Conclusion:: Despite this, three intratumoral injections of a plasmid-polyethylenimine complex resulted in substantial growth retardation of a poorly transfectable D2F2/E2 tumor in mice. There were no significant differences in anti-tumor properties between DNA constructs with telomerase or CAG promoters in vivo.

UCNP-based Photoluminescent Nanomedicines for Targeted Imaging and Theranostics of Cancer

Theranostic approach is currently among the fastest growing trends in cancer treatment. It implies the creation of multifunctional agents for simultaneous precise diagnosis and targeted impact on tumor cells. A new type of theranostic complexes was created based on NaYF4: Yb,Tm upconversion nanoparticles coated with polyethylene glycol and functionalized with the HER2-specific recombinant targeted toxin DARPin-LoPE. The obtained agents bind to HER2-overexpressing human breast adenocarcinoma cells and demonstrate selective cytotoxicity against this type of cancer cells. Using fluorescent human breast adenocarcinoma xenograft models, the possibility of intravital visualization of the UCNP-based complexes biodistribution and accumulation in tumor was demonstrated.

HER2-Specific Targeted Toxin DARPin-LoPE: Immunogenicity and Antitumor Effect on Intraperitoneal Ovarian Cancer Xenograft Model

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

Penetration Efficiency of Antitumor Agents in Ovarian Cancer Spheroids: The Case of Recombinant Targeted Toxin DARPin-LoPE and the Chemotherapy Drug, Doxorubicin

The efficiency of delivering a therapeutic agent into a tumor is among the crucial factors determining the prospects for its clinical use. This problem is particularly acute in the case of targeted antitumor agents since many of them are high-molecular-weight compounds. In this work, the penetration of therapeutic agents of two distinct molecular weights into the spheroids of ovarian adenocarcinoma overexpressing human epidermal growth factor receptor 2 (HER2) was studied. It was shown that the low-molecular-weight chemotherapy drug, doxorubicin (~0.5 kDa), effectively penetrates through almost the entire depth of a 300 to 400 μm spheroid, while the penetration depth of the HER2-specific recombinant targeted toxin, DARPin-LoPE (~42 kDa), is only a few surface layers of cells and does not exceed 70 μm. The low penetration of the targeted toxin into spheroid was shown along with a significant decrease in its efficiency against the three-dimensional tumor spheroid as compared with the two-dimensional monolayer culture. The approaches to increasing the accumulation of agents in the tumor are presented and prospects of their use in order to improve the effectiveness of therapy are discussed.

The Effect of the Targeted Recombinant Toxin DARPin-PE40 on the Dynamics of HER2-Positive Tumor Growth

The development of targeted toxins based on non-immunoglobulin targeting molecules appears to be one of the most advanced approaches in the targeted therapy of malignant tumors with a high expression of the HER2 receptor. Earlier, we showed that the targeted toxin DARPin-PE40 consisting of the HER2-specific non-immunoglobulin polypeptide (the targeting module) and a fragment of Pseudomonas exotoxin A (the toxic module) exhibits an antitumor effect in vivo against the HER2-positive adenocarcinoma xenograft. In this work, an in-depth analysis of the effect of DARPin-PE40 on the growth dynamics of experimental xenograft tumors was carried out. DARPin-PE40 was shown to inhibit tumor growth at a dose of 25 and 50 g/animal and to cause tumor node reduction at a dose of 80 g/animal, followed by growth resumption at the end of therapy. An evaluation of the tumor growth dynamics revealed statistically significant differences in tumor volume in mice in the experimental groups compared to the control group. The results testify to the potential of using the created targeted toxin as an agent for the targeted therapy of HER2-overexpressing tumors.